Compositions and methods for treating retinal degradation

ABSTRACT

The present disclosure relates to compositions and methods for treating retinal damage and/or retinal degradation. More specifically, this disclosure relates to methods for treating degradation of the retinal pigment epithelium by administering compositions comprising a nucleoside and/or a nucleoside or nucleotide reverse transcriptase inhibitor.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/861,290, filed Aug. 1, 2013, and from U.S. ProvisionalApplication Ser. No. 61/987,612, filed May 2, 2014, the entiredisclosures of which are incorporated herein by this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to compositions fortreating retinal damage and/or degradation. More specifically, thisdisclosure relates to methods for treating degradation of the retinalpigment epithelium by administering compositions comprising a nucleosideand/or a nucleoside reverse transcriptase inhibitor (NRTI).

BACKGROUND

Geographic atrophy, an advanced form of age-related macular degenerationthat causes blindness in millions of people worldwide and for whichthere is no approved treatment, results from death of retinal pigmentedepithelium (RPE) cells. For example, expression of DICER, an enzymeinvolved in microRNA (miRNA) biogenesis, is reduced in the RPE of humaneyes with geographic atrophy, and that conditional ablation of Dicer1induces RPE degeneration in mice. Surprisingly, ablation of seven otherenzymes responsible for miRNA biogenesis or function does not inducesuch pathology. Instead, knockdown of DICER1 leads to accumulation ofAlu repeat RNA in human RPE cells and of B1 and B2 (Alu-like elements)repeat RNAs in the RPE of mice. Alu RNA is dramatically increased in theRPE of human eyes with geographic atrophy, and introduction of thispathological RNA induces death of human RPE cells and RPE degenerationin mice.

Age-related macular degeneration (AMD), which is as prevalent as cancerin industrialized countries, is a leading cause of blindness worldwide.In contrast to the neovascular form of AMD, for which many approvedtreatments exist, the far more common atrophic form of AMD remainspoorly understood and without effective clinical intervention. Extensiveatrophy of the retinal pigment epithelium leads to severe vision lossand is termed geographic atrophy.

Hence, there remains a need for compositions and methods for treatingretinal degradation, and particularly RPE degradation.

BRIEF SUMMARY

This summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this summary does not list or suggest all possiblecombinations of features.

The present disclosure provides, in certain embodiments, a method fortreating retinal damage and/or degradation, comprising administering aneffective amount of a composition to a subject in need thereof, whereinthe composition comprises a reverse transcriptase inhibitor, such as anucleoside reverse transcriptase inhibitor (NRTI), selected from: (i) acompound having the structure of

or a pharmaceutically acceptable salt thereof;(ii) a compound having the structure of

or a pharmaceutically acceptable salt thereof; (iii) stavudine (d4T);(iv) lamivudine (3TC); (v) cordycepin; (vi) azidothymidine (AZT); (vii)abacavir (ABC); and/or (viii) a combination thereof.

Moreover, the methods of the present disclosure may further comprise thesteps of (i) inhibiting inflammasome activation by Alu RNA; (ii)reducing ATP-induced permeability of a cell; (iii) reducing an amount ofmitochondrial reactive oxygen species in a cell; and/or (iv) inhibitingactivation of at least one inflammasome in a subject's eye.Additionally, the cell(s) of the methods of the present disclosure maybe chosen, for example, from a retinal pigmented epithelium cell, aretinal photoreceptor cell, a choroidal cell, and a combination thereof.And an inflammasome of the present disclosure may be, for example, anNLRP3 inflammasome, an IL-1 beta inflammasome, or a combination thereof.

Furthermore, in some embodiments, the present disclosure provides acompound having the structure:

or a pharmaceutically acceptable salt thereof;or a compound having the structure

or a pharmaceutically acceptable sale thereof.

The present disclosure also provides a pharmaceutical compositioncomprising at least one of the compounds provided in the presentdisclosure, together with a pharmaceutically acceptable carrier. Andfurther embodiments of the present disclosure include a method forsynthesizing at least one compound provided in the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays a top row of ocular fundus photographs of mice receivingcontrol PBS, or Alu RNA treatment, with or without increasing amounts ofd4T (left to right); and RPE flat mounts, stained for intercellularjunctions (ZO-1) in red that are disrupted upon Alu RNA administrationbut that are restored to healthy RPE morphology/intercellular junctionsat highest dose of d4T.

FIG. 2 provides a bar graph showing that human (HeLa) cells treated withan enforced expression plasmid for Alu RNA (pAluA) for denoted amountsof time exhibited profoundly reduced viability compared to a nullplasmid (pUC19), as monitored by MTS proliferation assay and that d4Tco-administration prevented cell death induced by Alu overexpression.

FIG. 3 shows the results of Northern blotting using an Alu-specificprobe. As presented in FIG. 3, primary human RPE cells treated withantisense oligonucleotides targeting DICER1 (Dcr as) (lane 3 (third lanefrom left)) show increased Alu RNA levels in the nuclear compartmentcompared to control antisense oligonucleotides (Ctr as) (lane 1(leftmost)), and co-administration of d4T (lanes 2 and 4) does notreduce Alu RNA levels. u6 (bottom row) is shown as a loading control fornuclear fraction.

FIG. 4 provides another example of the results of Northern blottingusing an Alu-specific probe. As presented in FIG. 4, co-administrationof d4T does not change Alu RNA levels at 1, 4, or 24 hours aftertransfection in the nuclear fraction of human RPE cells transfected withAlu RNA, with or without d4T, as detected by Northern blotting using anAlu-specific probe. u6 (bottom row) is shown as loading control fornuclear fraction in FIG. 4.

FIG. 5 provides the results of a Western blot showing that Alu RNAcauses Caspase-1 maturation in primary human RPE cells at 24 hours afterAlu administration (top, middle lane, lower band), which is blocked byco-treatment with d4T (100 uM; rightmost lane). The bottom row is avinculin loading control.

FIG. 6 is a Western blot showing that Alu RNA causes Caspase-1maturation in primary human RPE cells at 24 hours after Aluadministration (top, middle lane, lower band), which is blocked withco-treatment with 3TC (20-100 uM; rightmost lane), wherein the lowermostband is the loading control, vinculin.

FIG. 7 is a Western blot showing that Alu RNA causes Caspase-1maturation in primary human RPE cells at 24 hours after Aluadministration (top, middle lane, lower band), which is blocked withco-treatment with azidothymidine (AZT), cordycepin, and abacavir (ABC)(50-100 uM; lanes 3-8 from left). The loading control vinculin is shownon the bottom.

FIG. 8 provides a gel showing that primary human RPE cells treated withLPS/ATP, a classic inflammasome activator, exhibit increased Casp-1activation, and phosphorylation of IRAK4, which is also a marker ofinflammasome signaling via the cell surface receptor adaptor proteinMyD88. Moreover, as shown in FIG. 8, d4T (25/100 uM) blocks Casp-1activation and IRAK4 phosphorylation induced by LPS/ATP. Vinculin wasused as the loading control in the gel of FIG. 8. Additionally, asshown, LPS and ATP activate the NLRP3 inflammasome only in combination.

FIG. 9 provides the results of Western blotting, wherein d4T, 3TC, andcordycepin (at 100 uM), all di-deoxy nucleoside reverse transcriptaseinhibitors, are shown to inhibit Caspase-1 activation (active p20 band,top) and IL-18 maturation (bottom) induced by LPS/ATP. To produce FIG.9, cell culture supernatants were collected after (i) no treatment, (ii)LPS treatment, or (iii) LPS/ATP treatment of mouse bone marrow-derivedmacrophages and run on Western blotting probing with antibodies forCaspase-1 and IL-18.

FIG. 10 provides the result of a Western blot showing that d4T (100, 250uM) inhibits IL-1 beta maturation (top, 18 and 22 kDa forms) andCaspase-1 activation (active p20 band, bottom) induced by nigericin. Toproduce FIG. 10, cell culture supernatants were collected after (i) notreatment, (ii) LPS treatment, or (iii) LPS/nigericin treatment of mousebone marrow-derived macrophages and run on Western blotting probing withantibodies for IL-1 beta and Caspase-1.

FIG. 11 shows a bar graph illustrating that d4T does not inhibit IL-1beta secretion from PMA-differentiated THP-1 monocytes induced bymonosodium urate (MSU). FIG. 11 was created after human THP-1 monocyteswere differentiated into macrophages with PMA, and, as shown in FIG. 11,treatment with MSU, a known inflammasome activator, increased IL-1 betasecretion compared to non-treated cells, whereas d4T co-administrationat a range of doses (25-1000 uM) did not significantly affect IL-1 betasecretion.

FIG. 12 is a bar graph, which shows that d4T and other nucleosidereverse transcriptase inhibitors do not inhibit IL-1 beta secretion fromPMA-differentiated THP-1 monocytes induced by MSU. Human THP-1 monocyteswere differentiated into macrophages with PMA. Their treatment with MSUincreased IL-1 beta secretion compared to non-treated cells, as shown inFIG. 12, while co-administration of d4T, 3TC, or cordycepin (all aredi-deoxy nucleotide analogs) at a range of doses (25-1000 uM) did notsignificantly affect IL-beta secretion.

FIG. 13 is a graph, which provides that d4T reduces NLRP3 priminginduced by Alu RNA. Indeed, as shown in FIG. 13, Alu RNA transfectionincreases NLRP3 mRNA levels in primary human RPE cells at 16 hours, anevent termed “priming” (Y-axis) compared to mock (transfection reagentalone). This effect is blunted by co-administration of d4T (100 uM) andnormalized to 18S RNA control.

FIG. 14 illustrates, in graph format, that Alu RNA transfectionincreases IL-1 beta mRNA levels in primary human RPE cells at 24 hours,an event termed “priming”, (Y-axis) compared to mock (transfectionreagent alone). This effect is blunted by co-administration of d4T (100uM) and normalized to 18S RNA control.

FIG. 15 shows that d4T reduces mitochondrial ROS caused by Aluexpression. Indeed, FIG. 15 demonstrates that enforced expression of Alu(pAluA) causes increased mitochondrial reactive oxygen species (mtROS),as detected by MitoSox assay. In order to produce FIG. 15, primary humanRPE cells were incubated with Alu expressing plasmid or control plasmid(pUC19) with or without d4T. After 15 hours cells were co-stained formtROS (red) and for cell count, nuclei (blue; Hoechst DNA stain). Cellsin the pAluA group exhibited greater mtROS staining (red) compared topUC19 control, an effect that is reduced in pAluA+d4T treated cells.

FIG. 16 provides a graph showing that d4T does not inhibit ATP releaseinduced by Alu RNA. Moreover, primary human RPE cells treated with AluRNA, for the times indicated in FIG. 16, release ATP. Cell culturesupernatant was collected from mock or Alu RNA treated cells, with orwithout d4T, and ATP was detected using an ATP-dependent luciferaseassay. Notably, d4T did not affect ATP release.

FIG. 17 shows that d4T reduces ATP-induced cell permeability to Yo-Pro1(P2X7 receptor assay). Indeed, d4T dose-dependently reduced Yo-Pro entryinduced by ATP, determined by an area-scan fluorescent measurement in a96 well microplate reader. FIG. 17 provides the results of thefluorescence measurement in relative fluorescence units (RFU, y-axis).

FIG. 18 illustrates, in graph format, that d4T reduces extracellularpotassium levels, which increase after Alu RNA transfection. Indeed,cell culture potassium levels increase in primary human RPE cellstreated with Alu RNA for 6 hours, an effect that is reduced by d4Tco-administration. Potassium levels were determined in cell culturesupernatants spectrophotometrically using a potassium-dependent pyruvatekinase assay.

FIG. 19 shows that d4T blocks bzATP-induced cell permeability to Yo-Pro1(P2X7 receptor assay). To prepare FIG. 19, d4T blocked YO-PRO-1 iodideentry in HEK293 cells stably expressing the human P2X7 receptorstimulated with the P2X7-selective agonist bzATP. Cells werepre-incubated with d4T for 30 minutes prior to addition of bzATP/YO-PRO,and fluorescence (in relative fluorescence units) at 485/515 nm wasmeasured at t=30 minutes.

FIG. 20 provides a chemical structure of methoxy-d4T (me-d4T). Morespecifically, as shown in FIG. 20, a single substitution of the ribose5′ hydroxyl group of d4T with a methoxy group (circled) has beendesigned to prevent d4T phosphorylation

FIG. 21 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA±me-d4T.

FIG. 22 shows cells, wherein unmodified d4T, but not me-d4T, blocksreplication of a GFP-expressing lentivirus in HeLa cells.

FIG. 23 provides a graph illustrating that unmodified d4T, but notme-d4T, reduces mtDNA levels (normalized to chromosomal DNA exon-intronjunction sequence) in primary mouse RPE cells as determined by real-timequantitative PCR. n=4, *p<0.05 by Student's t-test.

FIG. 24 provides flat mounts stained for zonula occludens-1 (ZO-1; red),bottom row. Degeneration outlined by blue arrowheads. Representativeimages of n=4 (B, C, E) shown. Scale bars, (C): 200 μm; (E): 20 μm

FIG. 25 provides a schematic overview of me-d4T synthesis.

FIG. 26 is an HPLC chromatogram of me-d4T (peak #6) final product, >97%purity.

FIG. 27 is a 1H NMR spectroscopy of me-d4T final product, wherein thechemical shifts are consistent with the structure of me-d4T.

FIG. 28 provides the results of liquid chromatography/mass spectrometryof me-d4T final product, m/z ratio consistent with the structure ofme-d4T.

FIG. 29 provides the methoxy variant of a nucleoside analog. Thechemical structure of 3TC (2′3′ dideoxycytidine) is shown, wherein themethoxy variation (O-methyl group) of nucleoside analog is circled.

FIG. 30 provides the methoxy variant of a nucleoside analog. Thechemical structure of AZT (3′-azido-2′,3′-dideoxythymidine) is shown,wherein the methoxy variation (O-methyl group) of nucleoside analog iscircled.

FIG. 31 provides the methoxy variant of a nucleoside analog. Thechemical structure of ABC (cyclopropylaminopurinylcyclopentene) isshown, wherein the methoxy variation (O-methyl group) of nucleosideanalog is circled.

FIG. 32 shows a cell permeant variant of d4T (IC-d4T), where “n” groupis equal to 11. Derivatives include cell permeant variants of 3TC, AZT,ABC, where the nucleobase group (circled) may be replaced, in variousembodiments, by 3TC, AZT, ABC, or methoxy-variants of d4T, 3TC, AZT, ABC(FIG. 29-31), or derivatives thereof.

FIG. 33 provides the structure of an exemplary NRTI according to thepresent disclosure.

FIG. 34 is a Western blot of Caspase-1 activation (p20 subunit) andIRAK4 phosphorylation in primary human RPE cells transfected with AluRNA±d4T.

FIG. 35 is a Western blot of Caspase-1 activation in human RPE cellstransfected with Alu RNA±NRTIs (3TC, AZT, ABC).

FIG. 36 includes fundus photographs: top row; flat mounts stained forzonula occludens-1 (ZO-1; red), bottom row. bars, 50 μm.

FIG. 37 provides fundus photographs: top row; flat mounts stained forzonula occludens-1 (ZO-1; red), bottom row. Scale bars, 50 μm.

FIG. 38 illustrates that NRTIs block LPS/ATP-induced inflammasomeactivation. Specifically, FIG. 38 shows a gel indicating that d4Tblocked Caspase-1.

FIG. 39 also illustrates that NRTIs block LPS/ATP-induced inflammasomeactivation, showing specifically a gel indicating that d4T blocked IL-1beta.

FIG. 40 presents chromatograms showing that Raji TK⁺ cells, but not RajiTK⁻ cells, phosphorylate AZT to AZT-triphosphate (AZT-TP) as determinedby liquid chromatography-mass spectrometry (LC-MS).

FIG. 41 shows that AZT blocks IL-1 beta activation by LPS/ATP in bothRaji TK⁻ and TK⁺ cells, as determined by Western blot of cell lysates.

FIG. 42 is a bar graph illustrating that d4T does not block Alu-inducedATP release from primary human RPE cells (n=4).

FIG. 43 provides a graph of P2X7-mediated YO-PRO-1 dye uptake(fluorescence) induced by bzATP (100 μM) in HEK293 cells stablyexpressing the human P2X7 receptor was inhibited by d4T and A438079 (64μM for both drugs). Fluorescence values are baseline subtracted fromcells without bzATP treatment. *bzATP vs. d4T; #bzATP vs. A438079,p<0.05 by Student-Newman Keuls post-hoc test (n=12).

FIG. 44 is a Western blot of Caspase-1 activation (p20 subunit) andIRAK4 phosphorylation in primary mouse RPE cells transfected with AluRNA±d4T.

FIG. 45 is a Northern blot of biotin-UTP-labeled Alu RNA-transfectedprimary human RPE cells.

FIG. 46 provides LC-MS/MS spectra of AZT-triphosphate (AZT-TP).

FIG. 47 provides LC-MS/MS spectra of AZU-triphosphate (AZT-TP

FIG. 48 shows the chromatographic separation of Raji TK⁻ cells spikedwith AZT-TP with MS spectra (inset) to confirm identity of designatedpeaks.

FIG. 49 shows the chromatographic separation of Raji TK⁻ cells spikedwith AZU-TP with MS spectra (inset) to confirm identity of designatedpeaks.

FIG. 50 is a standard curve of AZT-TP standards (black circle). Asshown, Raji TK⁺ samples treated with AZT produced AZT-TP (whitetriangles), whereas AZT-TP was not detectable in Raji TK⁻ cells treatedwith AZT.

FIG. 51 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA, with short peptide(Panx1¹⁰), which blocks P2X7 pore function but not cation flux (vs.scrambled peptide: Scr Panx1¹⁰).

FIG. 52 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA, with calmidazolium(FIG. 32 provides the chemical structure of IC- and EC-d4T used), whichblocks P2X7 cation flux but not pore function.

FIG. 53 is a Western blot of Caspase-1 activation (p20 subunit) inprimary human RPE cells transfected with Alu RNA, with cell permeable(IC), cell-impermeable (EC), or unmodified (no tag) d4T.

FIG. 54 shows that d4T prevents pAlu-induced mitochondrial ROSgeneration in primary human RPE cells. In FIG. 54, mitochondrialreactive oxygen species (ROS) were visualized with MitoSox (Red) andcell nuclei with Hoechst (Blue).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

The presently-disclosed subject matter is illustrated by specific butnon-limiting examples throughout this description. The examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the present invention(s). Each example is provided by way ofexplanation of the present disclosure and is not a limitation thereon.In fact, it will be apparent to those skilled in the art that variousmodifications and variations can be made to the teachings of the presentdisclosure without departing from the scope of the disclosure. Forinstance, features illustrated or described as part of one embodimentcan be used with another embodiment to yield a still further embodiment.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic(s) orlimitation(s) and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

While the following terms used herein are believed to be well understoodby one of ordinary skill in the art, definitions are set forth tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a fluorophore” includes aplurality of such images, and so forth.

Unless otherwise indicated, all numbers expressing quantities,properties, and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in this specification and claims are approximations that canvary depending upon the desired properties sought to be obtained by thepresently-disclosed subject matter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±50%, in someembodiments ±40%, in some embodiments ±30%, in some embodiments ±20%, insome embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%,in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed method.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The term “physiologically functional derivative” means anypharmaceutically acceptable derivative of a compound of the presentdisclosure. For example, an amide or ester of a compound of formula (I)or of a compound of formula (II), which upon administration to asubject, particularly a mammal, is capable of providing, either directlyor indirectly, a compound of the present disclosure of an activemetabolite thereof.

The terms “treatment” or “treating” refer to the medical management of asubject with the intent to cure, ameliorate, stabilize, or prevent acondition or disorder (e.g., retinal degradation). This term includesactive treatment, that is, treatment directed specifically toward theimprovement of a condition, and also includes causal treatment, that is,treatment directed toward removal of the cause of the associatedcondition. In addition, this term includes palliative treatment, thatis, treatment designed for the relief of symptoms rather than the curingof the condition; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development ofsymptoms or disorders of the associated condition; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder.

With regard to administering the compound, the term “administering”refers to any method of providing a composition and/or pharmaceuticalcomposition thereof to a subject. Such methods are well known to thoseskilled in the art and include, but are not limited to, oraladministration, transdermal administration, administration byinhalation, nasal administration, topical administration, intravaginaladministration, ophthalmic administration, intraaural administration,intracerebral administration, rectal administration, and parenteraladministration, including injectable such as intravenous administration,intra-arterial administration, intramuscular administration,subcutaneous administration, intravitreous administration, including viaintravitreous sustained drug delivery device, intracameral (intoanterior chamber) administration, suprachoroidal injection, subretinaladministration, Subconjunctival injection, sub-Tenon's administration,peribulbar administration, Transscleral drug delivery, administrationvia topical eye drops, and the like. Administration can be continuous orintermittent. In various aspects, a preparation can be administeredtherapeutically; that is, administered to treat an existing disease orcondition (e.g., exposure to OP compounds). In further various aspects,a preparation can be administered prophylactically; that is,administered for prevention of a disease or condition.

The term “effective amount” refers to an amount that is sufficient toachieve the desired result or to have an effect on an undesiredcondition. For example, a “therapeutically effective amount” refers toan amount that is sufficient to achieve the desired therapeutic resultor to have an effect on undesired symptoms, but is generallyinsufficient to cause adverse side effects. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

The terms “subject” or “subject in need thereof” refer to a target ofadministration, which optionally displays symptoms related to aparticular disease, condition, disorder, or the like. The subject(s) ofthe herein disclosed methods can be human or non-human (e.g., primate,horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, rodent, andnon-mammals). The term “subject” does not denote a particular age orsex. Thus, adult and newborn subjects, as well as fetuses, whether maleor female, are intended to be covered. The term “subject” includes humanand veterinary subjects.

As will be recognized by one of ordinary skill in the art, the terms“suppression,” “suppressing,” “suppressor,” “inhibition,” “inhibiting”or “inhibitor” do not refer to a complete elimination of angiogenesis inall cases. Rather, the skilled artisan will understand that the term“suppressing” or “inhibiting” refers to a reduction or decrease inangiogenesis. Such reduction or decrease can be determined relative to acontrol. In some embodiments, the reduction or decrease relative to acontrol can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.

In some exemplary embodiments, the presently-disclosed subject matterincludes methods for treating retinal damage and/or retinaldegeneration. Indeed, some methods of the present disclosure compriseadministering to a subject in need thereof an effective amount of acomposition for treating retinal damage and/or degradation.

In some embodiments the composition comprises a nucleoside and/or anucleoside reverse transcriptase inhibitor (NRTI). Further, in someembodiments, the composition is a pharmaceutical composition comprisinga nucleoside and/or a NRTI compound as well as a pharmaceuticallyacceptable carrier.

As discussed herein, in some exemplary methods of the presentdisclosure, the administered composition is a composition comprising anucleoside and/or NRTI. Thus, exemplary compositions are comprised ofcompounds including, but not limited to, stavudine (d4T), lamivudine(3TC), cordycepin, azidothymidine (AZT), abacavir (ABC), chemicalderivatives thereof (e.g., methoxy-derivatives to abrogatephosphorylation), and the like. Other possible compounds include, forexample, those described in U.S. Pat. No. 6,514,979 to Heredia et al.Those of ordinary skill in the art will also recognize furthernucleosides and/or NRTIs, as described herein, that can be used in thecompositions and methods of this disclosure.

In some embodiments a method of the present disclosure comprisesinhibiting activation of one or more physiological processes by Alu RNA.As disclosed herein, Alu RNA (including Alu repeat RNA in human cellsand B1 and B2, Alu-like element repeat RNAs) increases are associatedwith cells that are associated with certain conditions of interest. Forexample, an Alu RNA increase is associated with the retinal pigmentepithelium (RPE) cells of eyes with geographic atrophy. This increase ofAlu RNA induces the death of RPE cells. Methods and compositionsdisclosed herein can treat RPE degradation, thereby treating conditionsassociated with such cell death.

In some embodiments, a method of the present disclosure comprisesinhibiting the activation of at least one inflammasome. In certainembodiments, the at least one inflammasome is selected from an NLRP3inflammasome, a 1L-1 beta inflammasome, and a combination thereof. Insome embodiments, the inhibiting one or more inflammasomes of a cellincludes administering an inhibitor (composition) to the cell and/or toa subject, wherein the cell is the cell of a subject. For compositionscomprising an inhibitor, an inhibitor as described herein can be, forexample, a polypeptide inhibitor (including an oligonucleotideinhibitor), a small molecule inhibitor, and/or an siRNA inhibitor.

Moreover, some exemplary methods of administering the presentcomposition(s) can inhibit inflammation by LPS/ATP, inflammasomeactivation by LPS/ATP, inflammasome activation by Alu RNA, and/ornigericin-induced inflammasome activation. Exemplary methods can alsotreat retinal degradation and/or other retinal damage by reducingmitochondrial reactive oxygen species, particularly as caused by Alu RNAexpression, by blocking entry via the P2X7 receptor, and/or by reducingATP-induced cell permeability.

In some embodiments, a method of the present disclosure comprisestreating retinal damage by inhibiting a particular action in a cell. Insome embodiments, the cell is selected from an RPE cell, a retinalphotoreceptor cell, or a choroidal cell. In some embodiments, the cellis an RPE cell. In some embodiments, the cell is the cell of a subject.In some embodiments, the cell is a cell of a subject having, suspectedof having, or at risk of having a condition of interest. In someembodiments, the cell is a cell of a subject having, suspected ofhaving, or at risk of having age-related macular degeneration. In someembodiments, the cell is a cell of a subject having, suspected ofhaving, or at risk of having geographic atrophy. In some embodiments,the cell is a cell of a subject having, suspected of having, or at riskof having geographic atrophy and the cell is an RPE cell. In someembodiments, a subject having age-related macular degeneration can betreated using methods and compositions as disclosed herein.

Thus, as used herein with reference to a polypeptide being inhibited,“of a cell” refers to a polypeptide that is inside the cell (inside thecell membrane), on the cell (in the cell membrane, presented on the cellmembrane, otherwise on the cell), or outside of a cell, but insofar asthe polypeptide is outside of the cell, it is in the extracellularmilieu such that one of ordinary skill in the art would recognize thepolypeptide as being associated with the cell. For example, VDAC1,VDAC2, caspase-8, NFκB, or a polypeptide of an inflammasome (e.g.,NLRP3, PYCARD, caspase-1) could be in the cell. For another example,NLRP3 could be in the cell or on the cell.

As described herein, the presently-disclosed subject matter furtherincludes pharmaceutical compositions comprising the compounds describedherein together with a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” refers to sterile aqueousor nonaqueous solutions, dispersions, suspensions or emulsions, as wellas sterile powders for reconstitution into sterile injectable solutionsor dispersions just prior to use. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose.

Suitable formulations include aqueous and non-aqueous sterile injectionsolutions that can contain antioxidants, buffers, bacteriostats,bactericidal antibiotics and solutes that render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents.

The compositions can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods known in the art.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compositions can be formulated as eye drops. For example, thepharmaceutically acceptable carrier may comprise saline solution orother substances used to formulate eye drop, optionally with otheragents. Thus, eye drop formulations permit for topical administrationdirectly to the eye of a subject.

The compositions can also be formulated as a preparation forimplantation or injection. Thus, for example, the compounds can beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt). The compoundscan also be formulated in rectal compositions, creams or lotions, ortransdermal patches.

The presently-disclosed subject matter further includes a kit that caninclude a compound or pharmaceutical composition as described herein,packaged together with a device useful for administration of thecompound or composition. As will be recognized by those or ordinaryskill in the art, the appropriate administration-aiding device willdepend on the formulation of the compound or composition that isselected and/or the desired administration site. For example, if theformulation of the compound or composition is appropriate for injectionin a subject, the device could be a syringe. For another example, if thedesired administration site is cell culture media, the device could be asterile pipette.

Moreover, NRTIs of the present disclosure are a diverse, widely used,inexpensive class of small molecules, with extensive pharmacokinetic andsafety data collected over the past several decades of human use; NRTIsare therefore ripe for drug repurposing. As such, the present disclosureprovides a novel and broadly applicable basis for use of one or moreNRTIs by addressing major unmet medical needs.

As briefly described above, age-related macular degeneration is adisease that affects tens of millions of people worldwide, and there isno effective treatment for AMD (Ambati and Fowler, 2012). Similarly,graft-versus host disease is the major obstacle preventing successfultissue transplant (Ferrara et al., 2009); and sterile liver inflammationis a major contributor to drug-induced liver injury and steatohepatitis,a major determinant of fibrosis and carcinogenesis (Kubes and Mehal,2012). Thus, some methods and/or compounds of the present disclosure areintended to treat age-related macular degeneration, graft-versus hostdisease, and/or sterile liver inflammation by administering, in someembodiments, a compound comprising at least one NRTI, as provided in thepresent disclosure.

Since inflammasome inhibition by NRTIs can be achieved withoutphosphorylation of a particular NRTI, the use of me-d4T or otherphosphorylation-incompetent nucleoside analogs, as provided herein,should avoid therapeutic-limiting toxicities associated withNRTI-triphosphate-mediated polymerase inhibition (Lewis et al., 2003).Accordingly, in some embodiments, the present disclosure is directed tomethods for treating retinal disease by administering me-d4T or anotherphosphorylation-incompetent nucleoside analog to a subject in needthereof.

Further, in certain embodiments, the present disclosure provides methodsfor treating retinal damage, comprising: administering an effectiveamount of a composition to a subject in need thereof, wherein thecomposition comprises an NRTI. In some embodiments, the NRTI is selectedfrom the group consisting of stavudine (d4T), lamivudine (3TC),cordycepin, azidothymidire (AZT), abacavir (ABC), or derivatives orcombinations thereof.

In some embodiments, the presently disclosed subject matter providesmethods for protecting an RPE cell, a retinal photoreceptor cell, achoroidal cell, or a combination thereof comprising at least the step ofadministering a composition that comprises at least one nucleosideanalog or NRTI, according to the present disclosure, to a subject inneed thereof.

Moreover, in some embodiments, the present disclosure is directed to thesynthesis and/or use of one or more compounds of Formula I, Formula II,Formula III, Formula IV, and/or Formula IV:

and/or to any salt, particularly any pharmaceutically acceptable salt,any solvate, and/or any physiological derivative thereof. In someembodiments, “n” of Formula IV is any integer, and in a particularembodiment, n is 11.

Further, the present disclosure provides uses of a compound of any ofFormulas (I), (II), (III), (IV) and/or (IV), or any combination thereof,in the preparation or manufacture of a pharmaceutical composition, suchas a drug and/or medicine, especially a composition for the treatment ofretinal damage and/or retinal degeneration in a mammal. In someembodiments, the present disclosure provides a pharmaceuticalcomposition comprising a compound of any of Formulas (I), (II), (III),(IV) and/or (IV), any salt, particularly any pharmaceutically acceptablesalt, any solvate, and/or any physiological derivative thereof, togetherwith a pharmaceutically acceptable carrier.

In certain embodiments, the methods and compositions of the presentdisclosure inhibit graft-versus-host disease, chronic pain,proliferative vitreoretinopathy, glaucoma, rheumatoid arthritis,multiple sclerosis, bipolar disorder, major depressive disorder, renalfibrosis, nephritis, pulmonary fibrosis, Huntington's disease,osteoporosis, chronic lymphocytic leukemia, anxiety disorders, pulmonarytuberculosis, osteoporosis in post-menopausal women and fracturepatients, systemic lupus erythematosus, chronic inflammatory andneuropathic pain, autosomal dominant polycystic kidney disease, spinalcord injury, Alzheimer's disease, neuropathic pain, hypertension,varicose veins, type I diabetes, type II diabetes, gout, autoimmunehepatitis, graft vascular injury, atherosclerosis, thrombosis, metabolicsyndrome, salivary gland inflammation, traumatic brain injury, ischemicheart disease, ischemic stroke, Parkinson's disease, melanoma,neuroblastoma, prostate, breast, skin, and thyroid cancers, tubularearly gastric cancer, neuroendocrine cancer, mucoid colon cancer, coloncancer; high-grade urothelial carcinoma, kidney clear cell carcinoma,undifferentiated ovary carcinoma, papillary intracystic breastcarcinoma, gram negative sepsis, infectious Pseudomonas aeruginosa,Vibrio cholera, Legionella spp., Francisella spp., and Leishmania spp.Chlamydia spp., cryopyrinopathies; keratitis, acne vulgaris, Crohn'sdisease, ulcerative colitis, irritable bowel syndrome, insulinresistance, obesity, hemolytic-uremic syndrome, polyoma virus infection,immune complex renal disease, acute tubular injury, lupus nephritis,familial cold autoinflammatory syndrome, Muckle-Wells syndrome andneonatal onset multisystem inflammatory disease, chronic infantileneurologic cutaneous and articular autoinflammatory diseases, renalischemia-perfusion injury, glomerulonephritis, cryoglobulinemia,systemic vasculitides, IgA nephropathy, malaria, helminth parasites,septic shock, allergic asthma, hay fever, chronic obstructive pulmonarydisease, drug-induced lung inflammation, contact dermatitis, leprosy,Burkholderia cenocepacia infection, respiratory syncitial virusinfection, psoriasis, scleroderma, reactive arthritis, cystic fibrosis,syphilis, Sjögren's syndrome, inflammatory joint disease, non-alcoholicfatty liver disease, cardiac surgery (peri-/post-operativeinflammation), acute and chronic organ transplant rejection, acute andchronic bone marrow transplant rejection, tumor angiogenesis, and/or anycombination thereof.

Moreover, in some embodiments, the present disclosure provides thatnon-canonical NRTI function, independent of chain termination, preventsP2X7-dependent blindness, graft-versus-host disease and/or sterileinflammation. Accordingly, the present disclosure is directed, incertain embodiments, to methods of preventing P2X7-dependent blindness,graft-versus-host disease and/or inflammation in a subject byadministering an effective amount of at least one NRTI, as describedherein, to subject in need thereof.

Further, in certain embodiments, the methods and compositions of thepresent disclosure inhibit (i) inflammasome activation by Alu RNAassociated with a cell; (ii) inflammation by LPS/ATP, (iii) inflammasomeactivation by LPS/ATP, (iv) nigericin-induced inflammasome activation,and/or combinations thereof. And in some embodiments, the inflammasomeis selected from the group consisting of a NLRP3 inflammasome and/or a1L-1 beta inflammasome. Additionally, some embodiments of the methods ofthe present disclosure may include, for example, the steps of (i)blocking entry via a P2X7 receptor associated with a cell; (ii) reducingmitochondrial reactive oxygen species caused by Alu RNA expression;and/or (iii) reducing ATP-induced cell permeability of a cell. And acell contemplated in the present disclosure may include, for example, anRPE cell, a retinal photoreceptor cell, a choroidal cell, or anycombination thereof.

Further, NRTIs are mainstay therapeutics for HIV, and they blockretrovirus replication. Alu RNA, an endogenous retroelement that alsorequires reverse transcriptase (RT) for its life cycle, activates theNLRP3 inflammasome to cause cell death of the retinal pigment epitheliumin geographic atrophy, which is the untreatable form of age-relatedmacular degeneration that blinds millions of individuals. Moreover, theinventors of the present disclosure have found that NRTIs, as a class,are novel inhibitors of the NLRP3 inflammasome. And, surprisingly, thiseffect is independent of reverse transcriptase inhibition.

Thus, the inventors of the present disclosure have found that the NRTIsd4T, AZT, ABC, and 3TC block Caspase 1 activation by Alu RNA, as does5′-methoxy-d4T, which does not inhibit reverse transcriptase. Further,the present inventors have found that AZT is not phosphorylated inthymidine kinase-deficient cells but still blocks LPS/ATP-inducedinterleukin-1 beta secretion; that NRTIs block P2X7-dependent YOPRO-1dye uptake in mouse models of geographic atrophy, graft-versus-hostdisease, and sterile liver inflammation; and that NRTIs are novelinhibitors of the NLRP3 inflammasome independent of canonical reversetranscriptase inhibition. Accordingly, NRTIs are ripe for drugrepurposing in a variety of P2X7-driven diseases.

NRTIs were first discovered to be anti-viral compounds in 1974 (Ostertaget al., 1974), and are widely used to treat human immunodeficiency virus(HIV). The canonical mechanism of action of NRTIs is via chaintermination of DNA synthesis from a viral RNA template, therebyinterfering with the viral life cycle of reverse transcriptase-dependentviruses.

Age-related macular degeneration (AMD) is a leading cause of blindnessin the elderly worldwide (Ambati et al., 2003; Ambati and Fowler, 2012).In the more prevalent and untreatable dry form of AMD, overabundance ofnon-coding Alu RNAs causes blindness by inducing cell death of theretinal pigment epithelium (Dridi et al., 2012; Kaneko et al., 2011;Tarallo et al., 2012). Alu sequences are non-coding retrotransposonsthat, like HIV, rely on reverse transcriptase for their life cycle(Batzer and Deininger, 2002; Dewannieux et al., 2003).

Alu RNA mediates RPE cell death via activation of Caspase 1 and theNLRP3 inflammasome (Tarallo et al., 2012). The present disclosureprovides that a reverse transcriptase inhibitor, such as stavudine (d4T;2′3′ dideoxythymidine; Zerit, Bristol-Myers Squibb), which isFDA-approved for the treatment of HIV, prevents Caspase 1 cleavage toits active 20 kDa form (Hentze et al., 2003; Yamin et al., 1996) inprimary human (FIG. 34) and mouse RPE cells (FIG. 44) without reducingAlu RNA levels (FIG. 45). Further, the present disclosure shows that d4Talso blocks phosphorylation of IRAK4, a kinase downstream of the MyD88adaptor that mediates Alu-induced RPE cell death (Tarallo et al., 2012),in human and mouse RPE cells (FIG. 34 and FIG. 44). The inventors of thepresent disclosure have also found that other NRTIs, including theanti-HIV drugs azidothymidine (AZT; 3′-azido-2′,3′-dideoxythymidine;Retrovir, ViiV Healthcare), lamivudine (3TC; 2′3′ dideoxycytidine;Zeffix, GlaxoSmithKline) and abacavir (ABC; a di-deoxyguanosine analog;Ziagen, ViiV Healthcare), also block Caspase-1 cleavage induced by AluRNA (FIG. 35).

Additionally, the present disclosure provides that d4T and AZT preventRPE degeneration in the Alu RNA-induced mouse model of dry AMD. (Kanekoet al., 2011; Tarallo et al., 2012) Moreover, it has been found thatmice receiving daily oral administration of d4T blocked RPE degenerationafter sub-retinal injection of a plasmid expressing Alu RNA (FIG. 36),as did intraperitoneal administration of AZT (FIG. 37).

In order to test whether reverse transcriptase inhibition was requiredfor inflammasome blockade by d4T, a 5′ O-methyl-modified version of d4T(5′-OCH3-d4T; me-d4T) was synthesized (FIG. 20; FIG. 25, FIG. 36, FIG.27, FIG. 28). Accordingly, in some embodiments, the present disclosureis directed to methods for synthesizing a 5′ O-methyl-modified versionof d4T as provided herein.

Only the triphosphate version of nucleoside analogs inhibit reversetranscriptase; the methyl modification at the 5′ position preventsphosphorylation and thus formation of nucleoside triphosphate (Nykanenet al., 2001). Accordingly, like d4T, me-d4T also blocks Caspase-1activation in human RPE cells (FIG. 21).

The present inventors have confirmed that me-d4T does not inhibitreverse transcriptase: and, in contrast to unmodified d4T, me-d4T doesnot block lentivirus replication (FIG. 22). Also, the triphosphatemetabolite of di-deoxy nucleoside analogs caused depletion ofmitochondrial DNA; and consistent with the idea that me-d4T is notphosphorylated, it has been found that d4T, but not me-d4T reduces mtDNAlevels. (FIG. 23). Me-d4T also prevents Alu-induced RPE degeneration inmice (FIG. 24). These data indicate that d4T can block Caspase-1activation and RPE degeneration independent of reverse transcriptaseinhibition.

Further, the present inventors also tested whether NRTIs blockedinflammasome activation by LPS/ATP, which is not known to signal viareverse transcriptase (Mariathasan et al., 2004; Mariathasan et al.,2006; Martinon et al., 2002). It was found that d4T inhibitedLPS/ATP-induced Caspase-1 maturation in primary mouse bonemarrow-derived macrophages (FIG. 38) as detected by Western blot.

Caspase-1 directly processes interleukin 1 beta (IL-1 beta) upon LPS/ATPstimulation; d4T also blocks secretion of mature IL-1 beta in thesecells (FIG. 39). To determine whether LPS/ATP-induced inflammasomeactivation can be inhibited without RT inhibition, the present inventorsutilized thymidine kinase-deficient (Raji/TK⁻) and -expressing(Raji/TK⁺) cells (Balzarini et al., 1989). After addition of AZT, TK⁺,but not TK⁻ cells, the present inventors produced AZT-triphosphate(AZT-TP), the AZT metabolite required for RT inhibition (FIG. 40; FIG.46, FIG. 47, FIG. 48, FIG. 49, FIG. 50). Even though AZT was notphosphorylated in TK⁻ cells, AZT still inhibited LPS/ATP-inducedinterleukin-1 beta maturation (FIG. 41), indicating that AZT did notinhibit interleukin-1 beta maturation via reverse transcriptaseinhibition.

Alu RNA (Kerur et al., 2013) and LPS/ATP (Qu et al., 2011) activate theinflammasome via the ATP receptor P2X7. The present inventors thereforehypothesized that d4T blocks P2X7 or some P2X7-dependent pathway. First,testing was conducted to determine whether d4T acts upstream of P2X7 bymodulating ATP levels; however, d4T does not block release of ATP tocell culture media induced by Alu RNA (FIG. 42).

Next, testing was conducted to determine whether d4T directlyantagonizes P2X7 function: upon ATP binding, cell-surface P2X7 formsnon-selective cation channels that mediate inflammasome activation(Kahlenberg and Dubyak, 2004; Petrilli et al., 2007). However, d4T didnot significantly modulate P2X7 cation channel function as monitored bypatch clamp analysis of HEK293 stable cell lines expressing either thehuman or rat P2X7 receptor (Humphreys et al., 2000).

Finally, P2X7 activation is associated with the formation of a largepore that is permeable to molecules of up to ˜1000 Da (Adinolfi et al.,2005; Cheewatrakoolpong et al., 2005; Surprenant et al., 1996). It wasfound that d4T, and also AZT and 3TC, inhibited P2X7-dependent uptake ofthe fluorescent dye YO-PRO1 (M.W. Da) in human P2X7-overexpressingHEK293 stable cell line (FIG. 43) after addition of the selective P2X7agonist bzATP.

Consistent with the idea that NRTIs block Alu-induced P2X7-mediatedinflammasome activation via a mechanism involving dye uptake, AluRNA-induced Caspase-1 activation was inhibited by a small peptide thatblocks P2X7-mediated dye uptake and LPS/ATP-induced inflammasomeactivation, but not cation flux (Pelegrin and Surprenant, 2006) (FIG.51). On the other hand, Alu-induced Caspase-1 activation was notinhibited by calmidazolium, which selectively blocks P2X7-mediatedcation flux but not dye uptake (FIG. 52).

Furthermore, the intracellular C-terminus of P2X7 governsP2X7-associated dye uptake, and a version of d4T that is not cellpermeable (Agarwal et al., 2011) does not block caspase-1 activation byAlu RNA (FIG. 53, FIG. 32). Consistent with antagonism at or downstreamof P2X7, but upstream of mitochondrial dysfunction, d4T blocksmitochondrial ROS (mtROS) production, which are produced upon LPS/ATPstimulation (Adinolfi et al., 2005; Cruz et al., 2007; Garcia-Marcos etal., 2005; Nakahira et al., 2011) and Alu overexpression (Tarallo etal., 2012) was measured by MitoSOx assay (FIG. 54). Finally, d4T doesnot prevent P2X7-independent interleukin 1-beta secretion in PMA-primedTHP-1 cells treated with crystalline monosodium urate (FIG. 11)(Martinon et al., 2006; Riteau et al., 2012).

To explore the potential therapeutic relevance of NRTIs beyond theAlu-induced model of geographic atrophy (GA), it was hypothesized thatif NRTIs function as generic inflammasome inhibitors, then they might bebroadly useful in other animal models of disease that are also driven byP2X7. In the NLRP3 inflammasome- and P2X7-driven graft-versus-hostdisease model (Jankovic et al., 2013; Wilhelm et al., 2010), treatmentof mice receiving allogeneic bone marrow and T cells with d4T showedimproved survival compared to saline treated controls (30-70% vs. 0%).Furthermore, in the NLRP3- and P2X7-driven model of sterile inflammation(McDonald et al., 2010), d4T reduced neutrophil migration to the focusof liver injury.

Interestingly, it has been shown that P2X7-dependent pore function alonecan influence phenotype (Sorge et al., 2012). However, at present, thereare not any FDA-approved drugs that selectively target downstream P2X7signaling and not ion channel activation. Therefore, NRTIs could bevaluable both clinically and experimentally in the selective targetingof P2X7 function.

A role for P2X7 in regulating HIV replication was recently proposed(Hazleton et al., 2012), and HIV patients have increased plasma IL-18levels (Ahmad et al., 2002; Iannello et al., 2010), which decrease aftertreatment with NRTI-containing highly active anti-retroviral therapy(Stylianou et al., 2003). Notably, reduction of plasma IL-18 levels byNRTI treatment of HIV-1 infected patients did not significantlyassociate with viral load or CD4+ T-cell counts (David et al., 2000),indicating that NRTIs can dampen IL-18 levels before inhibition of viralreplication occurs. IL-18 maturation requires pro-IL18 cleavage byactive Caspase 1, which typically also requires P2X7 activation. Thus,the methods and experiments of the present disclosure are consistentwith the idea that NRTIs can modulate HIV-induced cytokine expressionindependent of reverse transcriptase inhibition.

In some embodiments, d4T prevents RPE degeneration induced by Alu RNA inwild type mice. As shown in FIG. 1, sub-retinal Alu RNA administrationto mice causes RPE degeneration in a mouse model of age-related maculardegeneration. Indeed, as shown, d4T co-delivered to the vitreous humorof wild type mice prevents Alu RNA-induced RPE cell death in adose-dependent manner at one week after delivery. The top row of FIG. 1provides an ocular fundus photograph of mice receiving control PBS, orAlu RNA treatment, with or without increasing amounts of d4T (left toright). Arrows denote depigmented regions of RPE cell death, whichresolve at highest dose of d4T. The bottom row of FIG. 1 shows an RPEflat mount, stained for intercellular junctions (ZO-1) in red that aredisrupted upon Alu RNA administration, but restored to healthy RPEmorphology/intercellular junctions at highest dose of d4T.

Meanwhile, in certain embodiments, d4T protects against cytotoxicityinduced by plasmid expressing Alu RNA in vitro. FIG. 2 shows that human(HeLa) cells treated with an enforced expression plasmid for Alu RNA(pAluA) for denoted amounts of time exhibited profoundly reducedviability compared to a null plasmid (pUC19), as monitored by MTSproliferation assay, and that d4T co-administration prevented cell deathinduced by Alu overexpression.

In some exemplary embodiments, d4T does not rescue cytotoxicity viareduction in Alu RNA levels. As presented in FIG. 3, primary human RPEcells treated with antisense oligonucleotides targeting DICER1 (Dcr as)(lane 3 (third lane from left)) show increased Alu RNA levels in thenuclear compartment compared to control antisense oligonucleotides (Ctras) (lane 1 (leftmost)), monitored by Northern blotting using anAlu-specific probe. Meanwhile, co-administration of d4T (lanes 2 and 4)does not reduce Alu RNA levels. FIG. 3 shows u6 (bottom row) as aloading control for nuclear fraction.

Moreover, in some embodiments, d4T does not reduce Alu RNA levels. Forexample, primary human RPE cells may be transfected with Alu RNA, withor without d4T. (FIG. 4) And, as presented in FIG. 4, co-administrationof d4T does not change Alu RNA levels at 1, 4, or 24 hours aftertransfection in the nuclear fraction, as detected by Northern blottingusing an Alu-specific probe. U6 (bottom row) is shown as loading controlfor nuclear fraction in FIG. 4.

The present disclosure further provides that, in some embodiments, d4Tinhibits inflammasome activation by Alu RNA. Indeed, Alu RNA causesNLRP3 inflammasome activation, which is marked by processing of theenzyme Caspase 1, and FIG. 5 provides a Western blot showing that AluRNA causes Caspase-1 maturation in primary human RPE cells at 24 hoursafter Alu administration (Top, Lane 2, lower band), which is blocked byco-treatment with d4T (100 uM; Lane 3). The bottom row in FIG. 5 is avinculin loading control.

In certain embodiments, 3TC inhibits inflammasome activation by Alu RNA.Indeed, Alu RNA causes NLRP3 inflammasome activation, which is marked byprocessing of the enzyme Caspase 1. FIG. 6 is a Western blot showingthat Alu RNA causes Caspase-1 maturation in primary human RPE cells at24 hours after Alu administration (top, lane 2, lower band), which isblocked with co-treatment with 3TC (20-100 uM; lane 3). On the bottom,the loading control, vinculin, is visible.

Next, FIG. 7 provides evidence of AZT, cordycepin, and abacavirinhibition of inflammasome activation by Alu RNA. Indeed, Alu RNA causesNLRP3 inflammasome activation, which is marked by processing of theenzyme Caspase 1. FIG. 7 is a Western blot showing that Alu RNA causesCaspase-1 maturation in primary human RPE cells at 24 hours after Aluadministration (top, lane 2, lower band), which is blocked withco-treatment with azidothymidine (AZT), cordycepin, and abacavir (ABC)(50-100 uM; Lanes 3-8). Again, the loading control vinculin is shown onthe bottom.

In certain embodiments, the present disclosure provides that d4Tinhibits inflammasome activation by LPS/ATP. As such, FIG. 8 provides agel showing that primary human RPE cells treated with LPS/ATP, a classicinflammasome activator, exhibit increased Casp-1 activation, andphosphorylation of IRAK4, which is also a marker of inflammasomesignaling via the cell surface receptor adaptor protein MyD88. Moreover,as shown in FIG. 8, d4T (25/100 uM) blocks Casp-1 activation and IRAK4phosphorylation induced by LPS/ATP. The loading control in FIG. 8 isvinculin. Furthermore, as shown, LPS and ATP activate the NLRP3inflammasome only in combination, thus treatment with one or the otheralone is useful as a control for this experiment.

The present disclosure further provides that, in exemplary embodiments,d4T and other NRTIs reduce inflammasome activation by LPS/ATP. Aspresented in FIG. 9, d4T, 3TC, and cordycepin (at 100 uM), all di-deoxynucleoside reverse transcriptase inhibitors, inhibit Caspase-1activation (active p20 band, top) and IL-18 maturation (bottom) inducedby LPS/ATP. To produce FIG. 9, cell culture supernatants were collectedafter (i) no treatment, (ii) LPS treatment, or (iii) LPS/ATP treatmentof mouse bone marrow-derived macrophages and run on Western blottingprobing with antibodies for Caspase-1 and IL-18.

In some embodiments of the present disclosure, d4T inhibitsnigericin-induced inflammasome activation. Per FIG. 10, d4T (100, 250uM) inhibits IL-1 beta maturation (top, 18 and 22 kDa forms) andCaspase-1 activation (active p20 band, bottom) induced by nigericin.Cell culture supernatants were collected after (i) no treatment, (ii)LPS treatment, or (iii) LPS/nigericin treatment of mouse bonemarrow-derived macrophages, and run on Western blotting probing withantibodies for IL-1 beta and Caspase-1. FIG. 10 shows the results ofthese efforts.

Additionally, in some embodiments, d4T does not inhibit IL-1 betasecretion from PMA-differentiated THP-1 monocytes induced by MSU. HumanTHP-1 monocytes were differentiated into macrophages with PMA. As shownin FIG. 11, treatment with monosodium urate (MSU), a known inflammasomeactivator, increased IL-1 beta secretion compared to non-treated cells,whereas d4T co-administration at a range of doses (25-1000 uM) did notsignificantly affect IL-1 beta secretion. Further, d4T does not blockMSU-induced IL-1 beta secretion as determined by ELISA (n=3-4).

In certain embodiments, d4T and other nucleoside reverse transcriptaseinhibitors do not inhibit IL-1 beta secretion from PMA-differentiatedTHP-1 monocytes induced by MSU. To illustrate this, human THP-1monocytes were differentiated into macrophages with PMA. Treatment withMSU increased IL-1 beta secretion compared to non-treated cells. (FIG.12) Meanwhile d4T, 3TC, or cordycepin (all are di-deoxy nucleotideanalogs) co-administration at a range of doses (25-1000 uM) did notsignificantly affect IL-1 beta secretion, as shown in FIG. 12.

Next, in some embodiments, d4T reduces NLRP3 priming induced by Alu RNA.Indeed, as provided in the bar graph of FIG. 13, Alu RNA transfectionincreases NLRP3 mRNA levels in primary human RPE cells at 16 hours, anevent termed “priming” (Y-axis) compared to mock (transfection reagentalone). This effect is blunted by co-administration of d4T (100 uM) andnormalized to 18S RNA control.

Furthermore, in exemplary embodiments of the present disclosure, d4Treduces IL-1 beta priming induced by Alu RNA. FIG. 14 illustrates thatAlu RNA transfection increases IL-1 beta mRNA levels in primary humanRPE cells at 24 hours, an event termed “priming”, (Y-axis) compared tomock (transfection reagent alone). This effect is blunted byco-administration of d4T (100 uM) and normalized to 18S RNA control.

Meanwhile, in some embodiments, d4T reduces mitochondrial ROS caused byAlu expression. FIG. 15 demonstrates that enforced expression of Alu(pAluA) causes increased mitochondrial reactive oxygen species (mtROS),as detected by MitoSox assay. In order to produce FIG. 15, primary humanRPE cells were incubated with Alu expressing plasmid or control plasmid(pUC19) with or without d4T. After 15 hours cells were co-stained formtROS (red) and for cell count, nuclei (blue; Hoechst DNA stain). Cellsin the pAluA group exhibited greater mtROS staining (red) compared topUC19 control, an effect that is reduced in pAluA+d4T treated cells.

And in further embodiments, d4T does not inhibit ATP release induced byAlu RNA. (FIG. 16) Primary human RPE cells treated with Alu RNA for thetimes indicated release ATP. To provide FIG. 16, cell culturesupernatant was collected from mock or Alu RNA treated cells, with orwithout d4T. ATP was detected using an ATP-dependent luciferase assay.And, notably, d4T did not affect ATP release.

In certain embodiments, d4T reduces ATP-induced cell permeability toYo-Pro1 (P2X7 receptor assay), as shown in FIG. 17. To prepare FIG. 17,THP-1 cells differentiated into macrophages by PMA allowed entry of thelarge fluorescent dye Yo-Pro 1, in an assay for P2X7 receptor activity.It was observed that d4T dose-dependently reduced Yo-Pro entry inducedby ATP, determined by an area-scan fluorescent measurement in a 96 wellmicroplate reader. Indeed, FIG. 17 provides the results of thefluorescence measurement in relative fluorescence units (RFU, y-axis).

Furthermore, it has been shown that d4T reduces extracellular potassiumlevels that increase after Alu RNA transfection. (FIG. 18) Indeed, cellculture potassium levels increase in primary human RPE cells treatedwith Alu RNA for 6 hours, an effect that is reduced by d4Tco-administration. For FIG. 18, potassium levels were determined in cellculture supernatants spectrophotometrically using a potassium-dependentpyruvate kinase assay.

Next, in some embodiments, d4T blocks bzATP-induced cell permeability toYo-Pro1 (P2X7 receptor assay), as shown in FIG. 19. d4T blocked YO-PRO-1iodide entry in HEK293 cells stably expressing the human P2X7 receptorstimulated with the P2X7-selective agonist bzATP. Cells werepre-incubated with d4T for 30 minutes prior to addition of bzATP/YO-PRO,and fluorescence at 485/515 nm measured at t=30 minutes.

Moreover, d4T blocks Alu-induced RPE degeneration and Caspase-1activation independent of reverse transcriptase inhibition.

In some embodiments, the present disclosure is directed to a compoundhaving the structure(s) provided in FIG. 20. FIG. 20 includes a chemicalstructure of methoxy-d4T (me-d4T) and of d4T. As shown in FIG. 20, asingle substitution of the ribose 5′ hydroxyl group with a methoxy group(circled) has been designed by the inventors of the present disclosureto prevent d4T phosphorylation. Accordingly, in some embodiments, thepresent disclosure is directed to a compound comprising a singlesubstitution of a ribose 5′ hydroxyl group with a methoxy group. And, insome embodiments, the present disclosure provides compounds comprising amethoxy group in place of a ribose 5′ hydroxyl group for preventingphosphorylation, such as d4T phosphorylation.

The present disclosure further provides the results of additionalexperiments in FIG. 21-FIG. 23. Indeed, FIG. 21 is a Western blot ofCaspase-1 activation (p20 subunit) in primary human RPE cellstransfected with Alu RNA±me-d4T; FIG. 22 shows cells, wherein unmodifiedd4T, but not me-d4T, blocks replication of a GFP-expressing lentivirusin HeLa cells; and FIG. 23 provides a graph illustrating that unmodifiedd4T, but not me-d4T, reduces mtDNA levels (normalized to chromosomal DNAexon-intron junction sequence) in primary mouse RPE cells as determinedby real-time quantitative PCR. n=4, *p<0.05 by Student's t-test.

In some embodiments, it has been shown that Me-d4T (intraperitonealinjection) prevents Alu-induced RPE degeneration in mice. FIG. 24, toprow, provides flat mounts stained for zonula occludens-1 (ZO-1; red),bottom row. Degeneration is outlined if FIG. 24 by blue arrowheads.Representative images of n=4 are shown.

Meanwhile, FIG. 25 provides a schematic overview of me-d4T synthesis,and FIG. 26 is an HPLC chromatogram of me-d4T (peak #6) finalproduct, >97% purity. And FIG. 27 is a 1H NMR spectroscopy of me-d4Tfinal product, wherein the chemical shifts are consistent with thestructure, and FIG. 28 provides the results of liquidchromatography/mass spectrometry of me-d4T final product, m/z ratioconsistent with the structure.

FIG. 29, FIG. 30 and FIG. 31 provide for methoxy variants of nucleosideanalogs. Specifically, FIG. 29 shows the chemical structure of 3TC (2′3′dideoxycytidine); FIG. 30 provides the chemical structure of AZT(3′-azido-2′,3′-dideoxythymidine); and FIG. 31 shows the chemicalstructure of ABC (cyclopropylaminopurinylcyclopentene). In each of FIGS.29-31, the methoxy variation (O-methyl group) of nucleoside analog iscircled. Further, FIG. 32 shows a cell permeant variant of d4T (IC-d4T),where “n” group is equal to 11. Derivatives include cell permeantvariants of 3TC, AZT, ABC, where the nucleobase group (circled) may bereplaced, in various embodiments, by 3TC, AZT, ABC, or methoxy-variantsof d4T, 3TC, AZT, ABC (FIG. 29-31), or derivatives thereof.

Meanwhile, FIG. 33 provides the chemical structure of an exemplary NRTIaccording to the present disclosure.

In certain embodiments, the present disclosure provides that NRTIs blockAlu-induced RPE degeneration and/or Caspase-1 activation. For example,FIG. 34 shows a Western blot of Caspase-1 activation (p20 subunit) andIRAK4 phosphorylation in primary human RPE cells transfected with AluRNA±d4T. FIG. 35 is a Western blot of Caspase-1 activation in human RPEcells transfected with Alu RNA±NRTIs (3TC, AZT, ABC). FIG. 36 shows thatpAlu causes RPE degeneration, which is prevented by oral administrationof d4T, and FIG. 37 shows that pAlu causes RPE degeneration, which isprevented by intraperitoneal administration of AZT. FIG. 36 and FIG. 37include fundus photographs: top row; flat mounts stained for zonulaoccludens-1 (ZO-1; red), bottom row. Degeneration is outlined by bluearrowheads. Scale bars, 50 μm.

FIGS. 38-41 illustrate that NRTIs block LPS/ATP-induced inflammasomeactivation. FIGS. 38 and 39 show that d4T blocked Caspase-1 (FIG. 38)and IL-1 beta (FIG. 39) activation in LPS/ATP treated primary mouse bonemarrow-derived macrophages as determined by western blot of cell culturemedia and lysate. Moreover, FIG. 40 presents chromatograms showing thatRaji TK⁺ cells, but not Raji TK⁻ cells, phosphorylate AZT toAZT-triphosphate (AZT-TP) as determined by liquid chromatography-massspectrometry (LC-MS). And FIG. 41 shows that AZT blocks IL-1 betaactivation by LPS/ATP in both Raji TK⁻ and TK⁺ cells as determined bywestern blot of cell lysates. Representative images of n=3-4 experimentsare provided in each of FIGS. 38-41.

In some embodiments, the present disclosure provides that NRTIsselectively block P2X7 pore function and P2X7-driven models of graftrejection and sterile liver inflammation, as shown in FIGS. 42-43. FIG.42 is a bar graph illustrating that d4T does not block Alu-induced ATPrelease from primary human RPE cells (n=4). Meanwhile, FIG. 43 is agraph illustration showing that NRTIs selectively block P2X7 porefunction and P2X7-driven models of graft rejection and sterile liverinflammation, providing a graph of the fluorescence (% of bzATP) overtime (minutes).

And in certain exemplary embodiments, the present disclosure providesthat d4T blocks Caspase-1 activation without reducing Alu RNA levels.Accordingly, FIG. 44 provides a

Western blot of Caspase-1 activation (p20 subunit) and IRAK4phosphorylation in primary mouse RPE cells transfected with Alu RNA±d4T.And FIG. 45 presents a Northern blot of biotin-UTP-labeled AluRNA-transfected primary human RPE cells. Notably, in FIG. 45, d4T didnot reduce Alu RNA levels (normalized to u6 RNA).

Next, FIGS. 46-47 provide LC-MS/MS spectra of AZT-triphosphate (AZT-TP,target compound; FIG. 46) and AZU-triphosphate (AZU-TP, internalstandard; FIG. 47). And FIGS. 48-49 show the chromatographic separationof Raji TK⁻ cells spiked with AZT-TP (FIG. 48) and AZU-TP (FIG. 49) withMS spectra (insets) to confirm identity of designated peaks.

FIG. 50 is a standard curve of AZT-TP standards (black circle). Raji TK⁺samples treated with AZT produced AZT-TP (white triangles), whereasAZT-TP was not detectable in Raji TK⁻ cells treated with AZT. FIG. 50 isrepresentative of two experiments.

FIGS. 51-54 show that, in some exemplary embodiments, P2X7-dependentpore function mediates Alu-induced Caspase-1 activation. Indeed, FIG. 51is a Western blot of Caspase-1 activation (p20 subunit) in primary humanRPE cells transfected with Alu RNA, with short peptide (Panx1¹⁰), whichblocks P2X7 pore function but not cation flux (vs. scrambled peptide:Scr Panx1¹⁰); FIG. 52 is a Western blot of Caspase-1 activation (p20subunit) in primary human RPE cells transfected with Alu RNA, withcalmidazolium (FIG. 32 provides the chemical structure of IC- and EC-d4Tused), which blocks P2X7 cation flux but not pore function; and FIG. 53is a Western blot of Caspase-1 activation (p20 subunit) in primary humanRPE cells transfected with Alu RNA, with cell permeable (IC),cell-impermeable (EC), or unmodified (no tag) d4T. Furthermore, FIG. 54shows that d4T prevents pAlu-induced mitochondrial ROS generation inprimary human RPE cells. In FIG. 54, mitochondrial reactive oxygenspecies (ROS) were visualized with MitoSox (Red) and cell nuclei withHoechst (Blue).

One of ordinary skill in the art will recognize that additionalembodiments or implementations are possible without departing from theteachings of the present disclosure or the scope of the claims whichfollow. This detailed description, and particularly the specific detailsof the exemplary embodiments and implementations disclosed herein, isgiven primarily for clarity of understanding, and no unnecessarylimitations are to be understood therefrom, for modifications willbecome obvious to those skilled in the art upon reading this disclosureand may be made without departing from the spirit or scope of theclaimed invention.

REFERENCES

Throughout this document various references are mentioned, includingpatent references. All such references are incorporated herein byreference, including the references set forth in the following list:

-   1. International Patent Application No. PCT/US11/38753.-   2. International Patent Application No. PCT/US12/46928.-   3. U.S. Provisional Patent Application Ser. No. 61/586,427.-   4. U.S. Provisional Patent Application Ser. No. 61/780,105.-   5. Adinolfi, E., Callegari, M. G., Ferrari, D., Bolognesi, C.,    Minelli, M., Wieckowski, M. R., Pinton, P., Rizzuto, R., and Di    Virgilio, F. (2005). Basal activation of the P2X7 ATP receptor    elevates mitochondrial calcium and potential, increases cellular ATP    levels, and promotes serum-independent growth. Mol Biol Cell 16,    3260-3272.-   6. Agarwal, H. K., Loethan, K., Mandal, D., Doncel, G. F., and    Parang, K. (2011). Synthesis and biological evaluation of fatty acyl    ester derivatives of 2′,3′-didehydro-2′,3′-dideoxythymidine. Bioorg    Med Chem Lett 21, 1917-1921.-   7. Ahmad, R., Sindhu, S. T., Toma, E., Morisset, R., and Ahmad, A.    (2002). Elevated levels of circulating interleukin-18 in human    immunodeficiency virus-infected individuals: role of peripheral    blood mononuclear cells and implications for AIDS pathogenesis. J    Virol 76, 12448-12456.-   8. Ambati, J., Ambati, B. K., Yoo, S. H., Ianchulev, S., and    Adamis, A. P. (2003). Age-related macular degeneration: etiology,    pathogenesis, and therapeutic strategies. Surv Ophthalmol 48,    257-293.-   9. Ambati, J., and Fowler, B. J. (2012). Mechanisms of age-related    macular degeneration. Neuron 75, 26-39.-   10. Balzarini, J., Herdewijn, P., and De Clercq, E. (1989).    Differential patterns of intracellular metabolism of    2′,3′-didehydro-2′,3′-dideoxythymidine and    3′-azido-2′,3′-dideoxythymidine, two potent anti-human    immunodeficiency virus compounds. J Biol Chem 264, 6127-6133.-   11. Batzer, M. A., and Deininger, P. L. (2002). Alu repeats and    human genomic diversity. Nat Rev Genet 3, 370-379.-   12. Cheewatrakoolpong, B., Gilchrest, H., Anthes, J. C., and    Greenfeder, S. (2005). Identification and characterization of splice    variants of the human P2X7 ATP channel. Biochem Biophys Res Commun    332, 17-27.-   13. Cruz, C. M., Rinna, A., Forman, H. J., Ventura, A. L.,    Persechini, P. M., and Ojcius, D. M. (2007). ATP activates a    reactive oxygen species-dependent oxidative stress response and    secretion of proinflammatory cytokines in macrophages. J Biol Chem    282, 2871-2879.-   14. David, D., Chevrier, D., Treilhou, M. P., Joussemet, M., Dupont,    B., Theze, J., and Guesdon, J. L. (2000). IL-18 underexpression    reduces IL-2 levels during HIV infection: a critical step towards    the faulty cell-mediated immunity? Aids 14, 2212-2214.-   15. Dewannieux, M., Esnault, C., and Heidmann, T. (2003).    LINE-mediated retrotransposition of marked Alu sequences. Nat Genet    35, 41-48.-   16. Dridi, S., Hirano, Y., Tarallo, V., Kim, Y., Fowler, B. J.,    Ambati, B. K., Bogdanovich, S., Chiodo, V. A., Hauswirth, W. W.,    Kugel, J. F., et al. (2012). ERK1/2 activation is a therapeutic    target in age-related macular degeneration. Proc Natl Acad Sci USA    109, 13781-13786.-   17. Ferrara, J. L., Levine, J. E., Reddy, P., and Holler, E. (2009).    Graft-versus-host disease. Lancet 373, 1550-1561.-   18. Garcia-Marcos, M., Fontanils, U., Aguirre, A., Pochet, S.,    Dehaye, J. P., and Marino, A. (2005). Role of sodium in    mitochondrial membrane depolarization induced by P2X7 receptor    activation in submandibular glands. FEBS Lett 579, 5407-5413.-   19. Hazleton, J. E., Berman, J. W., and Eugenin, E. A. (2012).    Purinergic receptors are required for HIV-1 infection of primary    human macrophages. J Immunol 188, 4488-4495.-   20. Hentze, H., Lin, X. Y., Choi, M. S., and Porter, A. G. (2003).    Critical role for cathepsin B in mediating caspase-1-dependent    interleukin-18 maturation and caspase-1-independent necrosis    triggered by the microbial toxin nigericin. Cell Death Differ 10,    956-968.-   21. Humphreys, B. D., Rice, J., Kertesy, S. B., and Dubyak, G. R.    (2000). Stress-activated protein kinase/JNK activation and apoptotic    induction by the macrophage P2X7 nucleotide receptor. J Biol Chem    275, 26792-26798.-   22. Iannello, A., Boulassel, M. R., Samarani, S., Tremblay, C.,    Toma, E., Routy, J. P., and Ahmad, A. (2010). HIV-1 causes an    imbalance in the production of interleukin-18 and its natural    antagonist in HIV-infected individuals: implications for enhanced    viral replication. J Infect Dis 201, 608-617.-   23. Jankovic, D., Ganesan, J., Bscheider, M., Stickel, N., Weber, F.    C., Guarda, G., Follo, M., Pfeifer, D., Tardivel, A., Ludigs, K., et    al. (2013). The Nlrp3 inflammasome regulates acute graft-versus-host    disease. J Exp Med 210, 1899-1910.-   24. Kahlenberg, J. M., and Dubyak, G. R. (2004). Mechanisms of    caspase-1 activation by P2X7 receptor-mediated K+ release. Am J    Physiol Cell Physiol 286, C1100-1108.-   25. Kaneko, H., Dridi, S., Tarallo, V., Gelfand, B. D., Fowler, B.    J., Cho, W. G., Kleinman, M. E., Ponicsan, S. L., Hauswirth, W. W.,    Chiodo, V. A., et al. (2011). DICER1 deficit induces Alu RNA    toxicity in age-related macular degeneration. Nature 471, 325-330.-   26. Kerur, N., Hirano, Y., Tarallo, V., Fowler, B. J.,    Bastos-Carvalho, A., Yasuma, T., Yasuma, R., Kim, Y., Hinton, D. R.,    Kirschning, C. J., et al. (2013). TLR-Independent and P2X7-Dependent    Signaling Mediate Alu RNA-Induced NLRP3 Inflammasome Activation in    Geographic Atrophy. Invest Ophthalmol Vis Sci 54, 7395-7401.-   27. Kubes, P., and Mehal, W. Z. (2012). Sterile inflammation in the    liver. Gastroenterology 143, 1158-1172.-   28. Lewis, W., Day, B. J., and Copeland, W. C. (2003). Mitochondrial    toxicity of NRTI antiviral drugs: an integrated cellular    perspective. Nat Rev Drug Discov 2, 812-822.-   29. Mariathasan, S., Newton, K., Monack, D. M., Vucic, D.,    French, D. M., Lee, W. P., Roose-Girma, M., Erickson, S., and    Dixit, V. M. (2004). Differential activation of the inflammasome by    caspase-1 adaptors ASC and Ipaf. Nature 430, 213-218.-   30. Mariathasan, S., Weiss, D. S., Newton, K., McBride, J.,    O'Rourke, K., Roose-Girma, M., Lee, W. P., Weinrauch, Y., Monack, D.    M., and Dixit, V. M. (2006). Cryopyrin activates the inflammasome in    response to toxins and ATP. Nature 440, 228-232.-   31. Martinon, F., Burns, K., and Tschopp, J. (2002). The    inflammasome: a molecular platform triggering activation of    inflammatory caspases and processing of proIL-beta. Mol Cell 10,    417-426.-   32. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., and    Tschopp, J. (2006). Gout-associated uric acid crystals activate the    NALP3 inflammasome. Nature 440, 237-241.-   33. McDonald, B., Pittman, K., Menezes, G. B., Hirota, S. A., Slaba,    I., Waterhouse, C. C., Beck, P. L., Muruve, D. A., and Kubes, P.    (2010). Intravascular danger signals guide neutrophils to sites of    sterile inflammation. Science 330, 362-366.-   34. Nakahira, K., Haspel, J. A., Rathinam, V. A., Lee, S. J.,    Dolinay, T., Lam, H. C., Englert, J. A., Rabinovitch, M., Cemadas,    M., Kim, H. P., et al. (2011). Autophagy proteins regulate innate    immune responses by inhibiting the release of mitochondrial DNA    mediated by the NALP3 inflammasome. Nat Immunol 12, 222-230.-   35. Nykanen, A., Haley, B., and Zamore, P. D. (2001). ATP    requirements and small interfering RNA structure in the RNA    interference pathway. Cell 107, 309-321.-   36. Ostertag, W., Roesler, G., Krieg, C. J., Kind, J., Cole, T.,    Crozier, T., Gaedicke, G., Steinheider, G., Kluge, N., and Dube, S.    (1974). Induction of endogenous virus and of thymidine kinase by    bromodeoxyuridine in cell cultures transformed by Friend virus. Proc    Natl Acad Sci USA 71, 4980-4985.-   37. Pelegrin, P., and Surprenant, A. (2006). Pannexin-1 mediates    large pore formation and interleukin-1 beta release by the ATP-gated    P2X7 receptor. Embo J 25, 5071-5082.-   38. Petrilli, V., Papin, S., Dostert, C., Mayor, A., Martinon, F.,    and Tschopp, J. (2007). Activation of the NALP3 inflammasome is    triggered by low intracellular potassium concentration. Cell Death    Differ 14, 1583-1589.-   39. Qu, Y., Misaghi, S., Newton, K., Gilmour, L. L., Louie, S.,    Cupp, J. E., Dubyak, G. R., Hackos, D., and Dixit, V. M. (2011).    Pannexin-1 is required for ATP release during apoptosis but not for    inflammasome activation. J Immunol 186, 6553-6561.-   40. Riteau, N., Baron, L., Villeret, B., Guillou, N., Savigny, F.,    Ryffel, B., Rassendren, F., Le Bert, M., Gombault, A., and    Couillin, I. (2012). ATP release and purinergic signaling: a common    pathway for particle-mediated inflammasome activation. Cell Death    Dis 3, e403.-   41. Sorge, R. E., Trang, T., Dorfman, R., Smith, S. B., Beggs, S.,    Ritchie, J., Austin, J. S., Zaykin, D. V., Vander Meulen, H.,    Costigan, M., et al. (2012). Genetically determined P2X7 receptor    pore formation regulates variability in chronic pain sensitivity.    Nat Med 18, 595-599.-   42. Stylianou, E., Bjerkeli, V., Yndestad, A., Heggelund, L.,    Waehre, T., Damas, J. K., Aukrust, P., and Froland, S. S. (2003).    Raised serum levels of interleukin-18 is associated with disease    progression and may contribute to virological treatment failure in    HIV-1-infected patients. Clin Exp Immunol 132, 462-466.-   43. Surprenant, A., Rassendren, F., Kawashima, E., North, R. A., and    Buell, G. (1996). The cytolytic P2Z receptor for extracellular ATP    identified as a P2X receptor (P2X7). Science 272, 735-738.-   44. Tarallo, V., Hirano, Y., Gelfand, B. D., Dridi, S., Kerur, N.,    Kim, Y., Cho, W. G., Kaneko, H., Fowler, B. J., Bogdanovich, S., et    al. (2012). DICER1 Loss and Alu RNA Induce Age-Related Macular    Degeneration via the NLRP3 Inflammasome and MyD88. Cell 149,    847-859.-   45. Wilhelm, K., Ganesan, J., Muller, T., Durr, C., Grimm, M.,    Beilhack, A., Krempl, C. D., Sorichter, S., Gerlach, U. V., Juttner,    E., et al. (2010). Graft-versus-host disease is enhanced by    extracellular ATP activating P2X7R. Nat Med 16, 1434-1438.-   46. Yamin, T. T., Ayala, J. M., and Miller, D. K. (1996). Activation    of the native 45-kDa precursor form of interleukin-1-converting    enzyme. J Biol Chem 271, 13273-13282.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A method for treating retinal degeneration,comprising: administering an effective amount of a composition to asubject in need of treatment for retinal degeneration, wherein thecomposition comprises a reverse transcriptase inhibitor selected from:(i) a compound having the structure of

 or a pharmaceutically acceptable salt thereof; (ii) a compound havingthe structure of

 or a pharmaceutically acceptable salt thereof; (iii) stavudine (d4T);(iv) lamivudine (3TC); (v) cordycepin; (vi) azidothymidine (AZT); (vii)abacavir (ABC); and (viii) a combination thereof.
 2. The method of claim1, comprising inhibiting inflammasome activation by Alu RNA associatedwith a cell.
 3. The method of claim 2, wherein the cell is a retinalpigmented epithelium cell, a retinal photoreceptor cell, a choroidalcell, or a combination thereof.
 4. The method of claim 1, comprisingreducing ATP-induced permeability of a cell.
 5. The method of claim 4,wherein the cell is a retinal pigmented epithelium cell, a retinalphotoreceptor cell, a choroidal cell, or a combination thereof.
 6. Themethod of claim 1, comprising reducing an amount of mitochondrialreactive oxygen species in a cell.
 7. The method of claim 1, comprisinginhibiting activation of at least one inflammasome in a subject's eye.8. The method of claim 7, wherein the at least one inflammasome isselected from an NLRP3 inflammasome, an IL-1 beta inflammasome, and acombination thereof.
 9. The method of claim 1, wherein the compositioncomprises a pharmaceutically acceptable carrier.
 10. A compound havingthe structure

or a pharmaceutically acceptable salt thereof.
 11. A pharmaceuticalcomposition comprising the compound of claim 10 and a pharmaceuticallyacceptable carrier.
 12. A compound having the structure

or a pharmaceutically acceptable salt thereof.
 13. A pharmaceuticalcomposition comprising the compound of claim 12 and a pharmaceuticallyacceptable carrier.
 14. A method for treating retinal degeneration,comprising: administering an effective amount of a composition to asubject in need of treatment for retinal degeneration, wherein thecomposition comprises: (i) a compound having the structure of

 or a pharmaceutically acceptable salt thereof; or (ii) a compoundhaving the structure of

 or a pharmaceutically acceptable salt thereof.
 15. The method of claim14, wherein the composition further comprises: stavudine (d4T),lamivudine (3TC), cordycepin, azidothymidine (AZT), abacavir (ABC), or acombination thereof.
 16. The method of claim 14, comprising inhibitinginflammasome activation by Alu RNA associated with a cell.
 17. Themethod of claim 16, wherein the cell is a retinal pigmented epitheliumcell, a retinal photoreceptor cell, a choroidal cell, or a combinationthereof.
 18. The method of claim 14, comprising reducing ATP-inducedpermeability of a cell.
 19. The method of claim 18, wherein the cell isa retinal pigmented epithelium cell, a retinal photoreceptor cell, achoroidal cell, or a combination thereof.
 20. The method of claim 14,comprising reducing an amount of mitochondrial reactive oxygen speciesin a cell.
 21. The method of claim 14, comprising inhibiting activationof at least one inflammasome in a subject's eye.
 22. The method of claim21, wherein the at least one inflammasome is selected from an NLRP3inflammasome, an IL-1 beta inflammasome, and a combination thereof. 23.The method of claim 14, wherein the composition comprises apharmaceutically acceptable carrier.